Strategy for Leukemia Therapy

ABSTRACT

The chimeric Bcr-Abl oncoprotein is the molecular hallmark of chronic myelogenous leukemia (CML). In the cytoplasm, the protein transduces a growth signal that is responsible for overexpansion of cells. In the nucleus, the protein induces apoptosis. The invention is a method of treating cancer/killing Bcr-Abl expressing cells by inducing the translocation of Bcr-Abl to the nucleus to activate the apoptotic pathway in cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 10/312,918 filed Dec. 27, 2002, which is a national stage filing under 35 U.S.C. 371 of International Application Number PCT/US01/20602 filed Jun. 29, 2001, which claims priority from U.S. Provisional Application Ser. No. 60/215,595 filed Jun. 30, 2000, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with government support from the National Institutes of Health under grant numbers CA 43054 and HL57900.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

The Sequence Listing, which is a part of the present disclosure, is a written sequence listing comprising nucleotide and amino acid sequences of the present invention. The written form of the Sequence Listing in the present application is identical to the computer readable form of the Sequence Listing in U.S. application Ser. No. 10/312,918 filed Dec. 27, 2002, from which this application claims priority. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Chronic myeloid leukemia (CML) is a hematological stem cell disorder characterized by excessive proliferation of cells of the myeloid lineage. The hallmark of CML is the Philadelphia chromosome, which arises from a reciprocal translocation between chromosomes 9 and 22 (Rowley, 1973). The molecular consequence of this translocation is the replacement of the first exon of c-Abl with sequences from the Bcr gene resulting in a Bcr-Abl fusion gene whose protein product shows enhanced tyrosine kinase activity (Bartram, et al., 1983; Ben-Neriah, et al., 1986; Heisterkamp et al., 1983; Konopka, et al., 1984; Shtivelman et al., 1985). The Bcr-Abl oncoprotein in CML is a 210-kD protein that contains 902 or 927 amino acids of Bcr fused to the expression product of exons 2-11 of c-Abl (Ben-Neriah, et al., 1986; Shtivelman et al., 1985). Found in 95% of patients with CML, p210 Bcr-Abl is also present in approximately 5-10% of adults with acute leukemia for whom there is no evidence of antecedent CML (Kruzrock, et al., 1988). Another Bcr-Abl fusion protein of 185 kD containing Bcr sequences from exon 1 (426 amino acids) fused to exons 2-11 of c-Abl, occurs in 10% of adult cases and 5-10% of pediatric cases of acute lymphoblastic leukemia (ALL), but not in CML (Clark, et al., 1988; Hermans et al., 1987). It is believed that this single chromosomal rearrangement is sufficient to initiate the development of these diseases and may be the only molecular abnormality in early stage disease.

Protein kinases are a large family of homologous proteins comprising 2 major subfamilies, the protein serine/threonine kinases and protein tyrosine kinases (PTKs). Protein kinases function as components of signal transduction pathways, playing a central role in diverse biological processes such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases with abnormalities in these signaling pathways is considered a promising approach for drug development. However, due to the structural and functional similarities of kinases, it is virtually impossible to make an inhibitor specific to a single kinase. Because of their deregulation in human cancers, Bcr-Abl, epidermal growth factor receptor (EGFR), HER2, and protein kinase C (PKC), were among the first protein kinases considered as targets for the development of selective inhibitors. As protein kinases have been implicated in more human cancers (Kolibaba and Drucker, 1997), drug-discovery efforts have been extended and several first-generation small-molecule inhibitors are now in various stages of development. A selection of these agents is shown in FIG. 1 (Drucker and Lyndon, 2000).

A kinase inhibitor, STI-571 (4-[(4-methyl-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyrodnyl)-2-pyrimidinyl]amin]phenyl]benzamide methanesulfonate; Glivec, Novartis, Basel, Switzerland) was initially identified in a screen for inhibitors of the platelet derived growth factor receptor (PDGFR) (Buchdunger et al., 1995). This ATP analog of the class 2-phenylaminopyrimidine, was found to have some selectivity for specific kinases including cdc2/cyclin B, c-FGR, protein kinase C γ and v-Abl. As the constitutive activation of v-Abl is believed to be sufficient for the development of CML, it was seen as an ideal target for validating the clinical utility of protein kinase inhibitors in the treatment of cancer.

The Abl oncogene was isolated originally from the genome of the Abelson murine leukemia virus (A-MuLV) (Rosenberg and Witte, 1988). This acutely transforming replication-defective virus encodes a transforming protein (p160v-Abl) with tyrosine-specific protein kinase activity. A-MuLV transforms fibroblasts in vitro and lymphoid cells in vitro and in vivo and was formed by recombination between Moloney murine leukemia virus (M-MuLV) and the murine c-Abl gene (Rosenberg and Witte, 1988).

As with many of the viral oncogenes, there are cellular equivalents that are involved in growth and differentiation. c-Abl is expressed normally in cells where it shuttles between the cytoplasm and the nucleus. This shuttling is driven by its three nuclear localization sequences (NLS) and one nuclear export sequence (NES). The NES is located in the C-terminus of c-Abl and contains a characteristic leucine rich motif. Mutation of a single leucine (L1064A) is sufficient to disrupt the function of the NES and result in the nuclear localization of c-Abl (Taagepera et al., 1998). Alternatively, the export of wild type c-Abl from the nucleus may be inhibited by the drug leptomycin B (LMB; NSC-364372D; PD114,720; elactocin), which functions by inactivating the nuclear export mediator CRM-1/exportin-1. The inactivation of the CRM-1 is irreversible and export is arrested until new protein is synthesized.

Cell adhesion and intracellular localization of c-Abl control the kinetics of its activation. c-Abl kinase activity is dependent on integrin mediated cell adhesion (Lewis et al, 1996). Upon cell adhesion, the cytoplasmic pool of c-Abl is reactivated within 5 minutes; however, the nuclear pool of c-Abl does not become reactivated for about 30 minutes, suggesting that activation occurs in the cytoplasm and the activated protein is translocated into the nucleus. In quiescent and G₁ cells, nuclear c-Abl is kept in an inactive state by the nuclear retinoblastoma protein (RB) that binds to the c-Abl tyrosine kinase domain and inhibits its activity. Phosphorylation of RB by the cyclin dependent kinases (CDKs) at the G₁/S boundary disrupts the RB-c-Abl complex, leading to activation of the c-Abl kinase. Nuclear c-Abl is part of the RB-E2F complex; thus, the G₁/S activation of c-Abl likely contributes to the regulation of genes involved in S-phase entry.

The nuclear c-Abl can be activated by DNA damage (Baskaran et al., 1997) to induce the p73 protein, which is a functional homolog of the tumor suppressor p53 (Gong, et al., 1999; Jost et al., 1997; Wang, 2000). Overexpression of p73 can activate the transcription of p53-responsive genes and inhibit cell growth in a p53-like manner by inducing apoptosis (Jost et al., 1997). Unlike c-Abl, Bcr-Abl is constitutively activated and is localized almost exclusively to the cytoplasm. The Bcr-Abl kinase activates a number of signal transduction pathways involved in cell proliferation and apoptosis (Warmuth, M., et al. 1999). For example, Bcr-Abl, which is typically cytoplasmic, can abrogate the dependence on interleukin-3 in hematopoietic cell lines (Daley et al., 1992) and the dependence on adhesion in fibroblastic cells (Renshaw et al., 1995). In addition, cytoplasmic Bcr-Abl can inhibit apoptosis through the activation of PI3-kinase, Akt and other mechanisms (Skorski et al., 1997; Amarante-Mendes et al., 1998). This mislocalization may be essential to the pathology of the protein.

STI 571 was shown to suppress the proliferation of Bcr-Abl-expressing cells in vitro and in vivo (Jost et al., 1999). In colony-forming assays of peripheral blood or bone marrow from patients with CML, STI 571 caused a 92-98% decrease in the number of Bcr-Abl colonies formed, with minimal inhibition of normal colony formation. (Jost et al., 1999) However, continual suppression of the Bcr-Abl tyrosine kinase is required for maximal clinical benefit in CML. Early studies demonstrated that Bcr-Abl-expressing cells could be rescued from apoptotic cell death if STI 571 were washed out of the cells within 16 hours of initial exposure (Drucker et al., 1996). If a tyrosine kinase inhibitor inhibited proliferation of Bcr-Abl-positive cells without inducing cell death, then long-term therapy would likely be required, suggesting that a well-tolerated, oral formulation of the drug would be needed.

Early pharmacokinetic studies in rats and dogs demonstrated that bioactive concentrations of STI571 are readily achieved in the circulation. However, the half-life of the drug is relatively short, 12-14 hours. In vivo STI571 treatment of nude mice injected with human leukemic cells was shown to eradicate 70-100% of the tumors. However, maintenance of a sufficiently high level of drug was essential for effective treatment. Mice receiving drug one or two times per day did not show significant improvements, whereas the mice receiving drug three times per day greatly improved (le Coutre et al., 1999). Additionally, relapse was seen in some animals which seemed to be mainly dependent on initial tumor load.

These pharmacokinetic properties make the drug less desirable for long term use. If a patient must take the drug long term, the chances of a patient maintaining a strict regimen of taking the drug on a strict interval decrease. No maximum tolerated dose has been identified for STI571 (Drucker, 2001a); however, its side effects are non-trivial for a drug that one must take in a regimented manner for an indefinite period of time. The most common side effects include nausea (in 43 percent of patients), myalgias (41 percent), edema (39 percent) and diarrhea (25 percent). Other side effects included fatigue, rash, dyspepsia, vomiting, theormbocytopenia, neutropenia and arthralgias.

Fluctuations in drug levels allow for the development of drug resistance which has been shown to happen in tissue culture (le Coutre et al., 2000). Treatment of LAMA84 human bcr-abl cells in culture with an initially sub-lethal, but continuously increasing dose of STI571 resulted in the development of a resistant cell line that was ten-fold more resistant to STI571 as compared to the parental cell line. Effectively, the process would be mimicked in a patient that did not adhere to a proper drug regimen. The cancer cells would be exposed to an insufficient dose of drug to induce quiescence or kill the more malignant cancer cells, allowing expansion of the resistant cells. Resistance develops by the amplification of the bcr-abl gene, a process which occurs during the blast crisis of the disease. Lack of adherence to the proper dosing regimen could hasten the onset of the blast crisis stage of the disease by promoting amplification of the gene.

Patients not responsive to standard interferon-a therapy were enrolled on a study to test the efficacy of STI571 as a chemotherapeutic agent (Drucker et al., 2001a). The study demonstrated that a relatively high level of drug must be maintained to have a therapeutic effect. Responses to STI571 were evaluated in two ways, hematologic, with complete response defined as a white-cell count of less than 10,000 per cubic millimeter; and cytogenetic, with a major response defined as less than 35 percent of the 20 metaphase cells analyzed positive for the Philadelphia chromosome. Hematologic responses were seen within two weeks, and all but one patient treated with 300 mg or more per day showed complete hematological response within four weeks. Only 31 percent of the patients had major cytogenetic responses, including 7 percent that showed complete cytogenetic remission. Cytogenetic responses occurred as early as two months and as late as 10 months. However, blood counts were maintained within normal limits regardless of whether a cytogenetic response was observed. Additionally, during treatment with STI571, blood counts gradually returned to normal during the first month, suggesting that the drug does not rapidly induce apoptosis, as would be expected with standard chemotherapy. Therefore, the drug seems to act by holding the diseased cells in abeyance rather than by killing them. This further emphasizes the need to maintain patients on STI571 indefinitely.

STI571 was also tested for efficacy in the treatment of patients who had entered the blast crisis of CML and acute lymphoblastic leukemia (ALL) (Drucker et al., 2001b). The blast crisis is highly refractory to treatment. The rate of response to standard induction chemotherapy is approximately 20 percent in myeloid blast crisis and 50 percent in lymphoid blast crisis. However, the remission seen is typically short lived. The blast crisis phase of both diseases is associated with genetic instability and multiple genetic abnormalities. Of the 58 patients enrolled in the study, 16 died due to disease progression. Other serious adverse events included nausea and vomiting, febrile neutropenia, elevated liver enzyme levels, exfoliative dermatitis, gastric hemorrhage, renal failure, pancytopenia and congestive heart failure. The improvements seen in some of the patients were short lived. Of the 38 patients with myeloid blast crisis, 4 had complete hematologic remission and 17 had a decrease in blasts in the marrow to less than 15 percent. Of these 21 that showed improvement, 9 subsequently relapsed between 42 and 194 days (median 84), 7 remained in remission with a follow-up of 101 to 349 days and three discontinued the study due to adverse events. Of the 14 patients with lymphoid blast crisis who had a response to STI571, 12 relapsed between 42 and 123 days (median 58). One patient remained in remission for 243 days of follow-up. The authors of the study stated that although Bcr-Abl plays a role in the blast crisis, inhibition of the single protein alone is insufficient to treat the disease and that combination with a second agent is required.

SUMMARY OF THE INVENTION

The invention is a method for the treatment of cancers expressing the bcr-abl oncogene product by localizing an active Bcr-Abl to the nucleus to induce apoptosis. Endogenous Bcr-Abl may be accumulated in the nucleus by use of drugs that alter the localization of the protein in the cancer cell by administration of a Bcr-Abl kinase inhibitor (e.g. STI571; PD173955 or PD180970 both from Parke-Davis) and leptomycin B (LMB) (Schaumberg et al., 1984). The kinase inhibitor encourages Bcr-Abl translocation into the nucleus. Functional block of the protein is reversed by clearance or breakdown of the kinase inhibitor while the nuclear localization of the protein is maintained by LMB. Upon reactivation of the nuclear pool of Bcr-Abl, apoptosis is induced. Such a therapeutic protocol can be used in vivo for the treatment of leukemias or ex vivo to purge bone marrow to allow for autologous bone marrow transplantation.

Alternatively the coding sequence for a mutant form of bcr-abl lacking a functional nuclear export signal (NES) (SEQ ID NO 1, 2) (Taagepera et al., 1998) can be transferred into a solid tumor by use of a nucleic acid expression cassette. Methods of transfer of “suicide genes” to solid tumors followed by treatment with drugs to kill the tumor cells is known to those skilled in the art (see U.S. Pat. No. 6,066,624, incorporated herein by reference). A number of gene transfer methods are known including, but are not limited to, adenoviral viral vectors, (e.g. U.S. Pat. No. 6,080,578, incorporated herein by reference), adenovirus associated vectors (e.g. U.S. Pat. No. 5,989,540, incorporated herein by reference) or retroviral vectors (e.g. U.S. Pat. No. 6,051,427, incorporated herein by reference). Alternatively, nucleic acid can be delivered by direct injection of DNA into tumor either alone or encapsulated in liposomes or other DNA transfer reagent (e.g. U.S. Pat. No. 5,976,567, incorporated herein by reference). Translocation of the NES-defective Bcr-Abl to the nucleus is driven by administration of a kinase inhibitor. Apoptosis is promoted in cells containing the nuclear localized Bcr-Abl by washout of the kinase inhibitor to allow for activation of the protein. The rapid clearance of STI571 allows for the activation of the nuclear Bcr-Abl which induces apoptosis.

In another embodiment, the coding sequence for the NES-defective Bcr-Abl is fused in frame to the coding sequence of at least one heterologous nuclear localization signal (NLS) (e.g. SV40 large T antigen, nucleoplasmin) for transfer into a solid tumor by any of the methods listed above. The presence of the NLS abrogates the need for therapy with a kinase inhibitor or LMB. Upon translation of the protein, it is translocated into the nucleus where it activates apoptotic pathways.

This method overcomes a number of the problems with the use of STI571 as a single chemotherapeutic agent. First, the cancer cells are killed and not simply maintained in a quiescent state. This overcomes difficulties of maintaining rigorous therapeutic schedules for indefinite periods of time, increasing the quality of life for patients as well as decreasing the chances of the development of resistance. Such a therapy can be useful in blast crisis as only a portion of the Bcr-Abl must be translocated into the nucleus to induce apoptosis. The severe adverse events seen in the study with patients in the blast crisis stage of the disease would not likely be seen with less than a week of drug treatment. Steady state blood levels of 1 μM STI571 and 10 nm LMB, effective doses of the drugs in culture, can be reached in two to three days. STI571 is rapidly cleared in a predictable manner with the time line dependent on the amount of drug given to the patient. If the Bcr-Abl is the endogenous protein from the cell, the patient is maintained on LMB for 2 to 3 days after combined treatment with a kinase inhibitor and LMB to retain the protein in the nucleus. Similar time considerations would be appropriate for LMB to purge bone marrow ex vivo. Ex vivo, effective doses of kinase inhibitor and LMB can be reached essentially instantaneously. In mouse bone marrow, clearance of at least 98% of the Bcr-Abl expressing bone marrow cells was achieved by treatment of the cells for 12 hours with STI571 with LMB added for the last 8 hours of STI571 treatment. Maintaining the cells in LMB alone for an additional period of time after treatment with the drugs together can increase the rate of cell killing as with the tissue culture cells. If the Bcr-Abl is a nuclear export defective mutant transferred to the tumor by gene therapy, the patient is treated with kinase inhibitor alone for up to a week to encourage the entry of the protein into the nucleus. If an NLS supplemented NES-defective Bcr-Abl construct is used, no treatment with drugs is required. Upon completion of the first round of chemotherapy, the patient is monitored and the regimen may be repeated as needed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Table of selected small molecule ATP-competitive protein kinase inhibitors in development.

FIG. 2. Nuclear localization of active Bcr-Abl kinase kills cells.

FIG. 3. Irreversible killing of Bcr-Abl transformed cells. The indicated cells were left untreated (◯), exposed to LMB (▪), STI571 (▴), or to both drugs (×) for 48 hours (Ton B) or 72 hours (K562). Cells were then washed, plated in fresh media, and viable cells were counted every two days. The results of one representative experiment from three independent experiments is shown. a, Ton B cells that are not induced to express Bcr-Abl. b, TonB210 cells cultivated in the absence of IL-3. c, TonB210 cells grown in the presence of IL-3. d, K562 cells.

FIG. 4. Preferential killing of mouse bone marrow cells expressing Bcr-Abl. a, Murine bone marrow cells were collected 5 days after 5-FU injection. Cells were infected with MSCV-Bcr-Abl/P210-IRES-GFP retrovirus. A mixed population of infected and uninfected cells was analyzed by FACS to determine the percentage of GFP⁺ cells before treatment (BT). The population was divided equally among 4 conditions: untreated (NT), incubated with LMB (L), STI571 (S), or STI571 plus LMB (S+L). After drug removal, cells were cultured in normal growth media and the collected for FACS analysis. The results shown are from a representative experiment performed in duplicate. b, The same experiment as in a was repeated with bone marrow cells from mice that were not injected with 5-FU. c, Total number of live cells from experiment in b were determined and normalized to the cell number of the mixed population before treatment. The uninfected (GFP⁻) and the infected (GFP⁺) cells were similarly sensitive to treatment with LMB or STI alone. The GFP⁺ cells were extremely sensitive to the combined treatment, whereas the GFP⁻ cells were not.

The present invention will be better understood from the following detailed description of an exemplary embodiment of the invention, taken in conjunction with the accompanying drawings in which like reference numerals refer to like parts and in which:

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

The cytoplasmic Bcr-Abl tyrosine kinase is a potent inhibitor of apoptosis (McGahon et al., 1994). The anti-apoptotic activity of Bcr-Abl contributes to the development of CML and the resistance of CML cells to chemotherapy (Warmuth et al., 1999). Evidence is presented that the Bcr-Abl tyrosine kinase can be converted into an activator of apoptosis by allowing it to function inside the nucleus. Apoptosis induced by the nuclear Bcr-Abl cannot be suppressed by the cytoplasmic Bcr-Abl, because nuclear entrapment of a fraction of the total Bcr-Abl is sufficient to kill cells. Because the nuclear Bcr-Abl can kill cells, cytoplasmic retention of this activated tyrosine kinase is required for cell transformation. That the Bcr-Abl kinase can induce apoptosis from the nucleus is consistent with the role of the nuclear c-Abl tyrosine kinase in the activation of cell death (Gong et al., 1999; Wang, 2000).

The discovery of STI571 has raised the prospect of a CML treatment with increased efficacy and limited side effects (Drucker and Lyndon, 2000). STI571 has shown efficacy in phase I and phase II clinical trials on CML patients in the chronic phase (Goldman, 2000). It is already evident, however, that STI571 does not have a long-term efficacy on CML patients in the acute phase (Vastag, 2000). Moreover, prolonged exposure to STI571, CML cells could develop resistance to this drug (Gambacourti, et al., 2000; le Coutre et al., 2000; Mahon et al., 2000; Weisberg and Griffin, 2000). LMB is a potent inhibitor of cell proliferation; however, its therapeutic application is limited by neuronal toxicity observed in a phase I clinical trial (Newlands et al., 1996). Though LMB is toxic to cells irrespective of Bcr-Abl expression, its effect is reversible after drug removal. Experiments on mouse bone marrow cells suggest that the combined treatment with LMB and STI571 is useful to purge explanted bone marrow of CML cells. This purging strategy allows autologous bone marrow transplantation to become a therapeutic option for CML.

Previous studies have shown that STI571 could induce apoptosis of CML cell lines in culture (Beran et al., 1998; Deininger et al., 1997). Herein is disclosed an alternative mechanism to induce apoptosis through STI571. When STI571 is combined with an inhibitor of nuclear export, it causes Bcr-Abl to be trapped in the nucleus. When the nuclear Bcr-Abl recovers its kinase activity, through the removal or metabolic decay of STI571, apoptosis is activated. These results indicate an interesting approach to treating CML, by trapping Bcr-Abl in the nucleus of leukemic cells and thus converting Bcr-Abl into a terminator of this disease.

Studies demonstrated that the kinase defective Bcr-Abl (Bcr-Abl-KD) entered the nucleus spontaneously; therefore, STI571 was tested to determine if it could stimulate the nuclear import of wild-type Bcr-Abl through inactivation of the kinase. Abl^(−/−) fibroblast were transfected using the calcium phosphate method, well known to those skilled in the art, with plasmids encoding Bcr-Abl. Cells were treated with STI571 alone or with a combination of STI571 and LMB. Treatment with STI571 alone did not result in the nuclear accumulation of Bcr-Abl. However, the combined treatment with STI571 and LMB led to the accumulation of Bcr-Abl in the nucleus.

To quantify the stimulatory effect of STI571 on the nuclear import of Bcr-Abl without the influence of LMB, the NES of Bcr-Abl was inactivated by mutating a critical leucine residue (L1064A) (SEQ ID NO 1, 2) (Taagepera, et al., 1998). The NES mutation affects neither the kinase activity nor the interaction of Bcr-Abl with STI571, which binds to the tyrosine kinase domain (Schindler et al., 2000). When expressed in Abl^(−/−) cells, BCR-Abl-NES was localized to the cytoplasm (FIG. 2 a). Following incubation with STI571, BCRAbl-NES became detectable in the nucleus (FIG. 2 b). STI571 did not alter the steady-state levels of BCR-Abl-NES (FIG. 2 c). The fraction of BCR-Abl-NES found in the nucleus was dependent on the concentration of STI571 and increased with time (FIG. 2 d). The amount of BCR-Abl-NES in the nucleus was 5, 10 and 20%, respectively, after a 12-hour incubation with 0.1, 1 and 10 M of STI571. Between 12 and 18 hours, the amount of nuclear Bcr-Abl NES continued to increase with 0.1 and 1 μM of STI571, but approached a plateau with 10 μM of STI571 (FIG. 2 d). The in vivo IC₅₀ for the inhibition of Bcr-Abl tyrosine kinase is 0.25-0.4 pM (Drucker et al., 1996). Thus, the nuclear accumulation of Bcr-Abl-NES could be correlated with the inhibition of its kinase activity. The nuclear fraction of Bcr-Abl-NES at 24 hours of treatment was between 25-35%. The nuclear fraction of Bcr-Abl-KD treated with LMB alone, or Bcr-Abl treated with both LMB and STI571 was also between 25-35%. These results indicate that mechanisms other than the kinase activity also mediate the cytoplasmic retention of Bcr-Abl.

It is known that activation of the nuclear Abl tyrosine kinase by DNA damage can contribute to the induction of apoptosis (Gong et al., 1999; Wang, 2000). Similarly, when translocated into the nucleus, Bcr-Abl kinase can activate apoptosis. The mere expression of Bcr-Abl does not cause apoptosis (as measured by TUNEL assay). However, trapping Bcr-Abl in the nucleus by combined treatment with STI571 and LMB caused apoptosis in 20% of Bcr-Abl expressing cells, which was not significantly different from the 10% of untransfected cells (Table 2). Maintenance of Bcr-Abl in the inactivated form did not stimulate apoptosis. However, removal of the inhibitor while maintaining the Bcr-Abl in the nucleus strongly stimulated apoptosis. Following incubation with both STI571 and LMB to trap Bcr-Abl in the nucleus, cells were washed extensively and placed in fresh media with LMB alone to allow the recovery of Bcr-Abl kinase activity in the nucleus. With this protocol, between 70-80% of the Bcr-Abl expressing cells were positive for TUNEL staining, whereas the apoptosis rate remained at the 10% level with untransfected cells (Table 2). By expressing Bcr-Abl-KD and treating cells with LMB alone, it was confirmed that an active nuclear Bcr-Abl kinase was required to induce apoptosis. Despite its nuclear accumulation, Bcr-Abl-KD did not induce apoptosis (Table 2).

The inhibition of nuclear export by LMB has a cytotoxic effect of its own. To rule out that Bcr-Abl-induced apoptosis is dependent entirely on LMB, BCR-Abl-NES, which could be trapped in the nucleus by treatment with STI571 alone was tested for its ability to induce apoptosis (Table 2). The nuclear accumulation and re-activation of BCR-Abl-NES was able to cause apoptosis, in that 60% of the BCR-Abl-NES expressing cells became TUNEL-positive as compared with only 3% of the untransfected cells (Table 2). This result demonstrates that the nuclear Bcr-Abl kinase induces apoptosis in the absence of LMB.

Response to the drug regimen was further tested in a CML cell line, K562, and a non-CML myeloid leukemic cell line, HL60. Treatment of cells with a combination of STI571 and LMB for 8 hours, followed by treatment of the cells with LMB alone for 4 hours induced apoptosis in the K562 cells, but not in the HL60 cell line, further confirming the role of Bcr-Abl in the killing of the cells. Additionally, the studies demonstrated that the constitutively expressed Bcr-Abl, rather than just transiently expressed Bcr-Abl, may participate in the cell killing process induced by STI571 and LMB.

STI571 is more effective in preventing cells from dividing than it is in killing them. Upon removal of the drug, cells are able to proliferate again. To determine the efficacy and the specificity of cell killing by the nuclear Bcr-Abl, the long-term survival of drug-treated cells was investigated. The murine pro-B cell line TonB, which can be induced to express p210 Bcr-Abl through a doxycycline-regulated promoter was used (Klucher et al., 1998). The uninduced TonB and the induced TonB210 (expressing Bcr-Abl) cells were treated for a total of 48 hours with either LMB or STI571 alone, or both drugs combined. At the end of the 48-hour period, cells were washed extensively, placed in fresh media and the number of live cells was counted every 48 hours for a total of 14 to 16 days (FIG. 3 a-c).

TonB cells that were not induced to express Bcr-Abl did not exhibit any reaction to STI571 (FIG. 3 a). Treatment of TonB cells with LMB alone or with LMB plus STI571 caused a reduction in cell numbers (FIG. 3 a). LMB causes an irreversible inactivation of Crml/exportin-1 (Kudo et al., 1999); however, cells can recover from LMB, through the de novo synthesis of Crml/exportin-1. Indeed, TonB cells resumed growth several days after the removal of LMB (compare FIG. 3 a). Because TonB cells are dependent on IL-3 for survival and proliferation (Klucher et al., 1998), IL-3 was included in these experiments.

TonB210 cells induced to express Bcr-Abl became IL-3-independent (Klucher et al., 1998). The drug treatments were performed on TonB210 cells with and without IL-3. Without IL-3, TonB210 cells were sensitive to treatment with STI571 (FIG. 3 b). Treatment with LMB alone for 48 hours also caused a decrease in cell numbers. Nevertheless, TonB210 cells could resume growth following the removal of LMB or STI571 (FIG. 3 b). In contrast, the combined treatment with STI571 and LMB for 48 hours caused the complete loss of viable cells after the removal of drugs (FIG. 3 b). With IL-3, TonB210 cells were not sensitive to treatment with STI571 (FIG. 3 c). The inclusion of IL-3 also promoted the recovery of TonB210 cells following the removal of LMB (compare FIGS. 3 b and c). However, even with the continuous presence of IL-3, treatment with a combination of STI571 and LMB caused a complete eradication of the TonB21 0 population following drug removal (FIG. 3 c). In combination with LMB, concentrations of STI571 between 1 to 10 μM were similarly effective in mediating the complete killing of TonB210.

Irreversible killing by the combined action of STI571 and LMB in the blast crisis cell line K562 was observed (FIG. 3 d). Similar to the murine TonB210 cells, K562 cells showed initial death when STI571 was present, but this was consistently followed by recovery after the removal of STI571 (FIG. 3 d). A similar initial decline in cell number was observed and recovery when K562 cells were treated with LMB alone (FIG. 3 d). Again, treatment with both STI571 and LMB resulted in the complete loss of viable K562 cells by six days after drug removal.

The precipitous decline in viable TonB210 or K562 cells occurred several days after the removal of both drugs (FIG. 3 b-d). One reason for this delayed killing could be the time required to recover the Bcr-Abl kinase activity in the nucleus. The tyrosine-phosphorylated proteins in total cell lysates were examined at the end of drug treatments and then before their precipitous death. TonB cells showed a low level of tyrosine phosphorylation, which was not affected by the drug treatments. In TonB210 and K562 cells, the overall levels of tyrosine-phosphorylated proteins decreased following incubation with STI571 alone or STI571 plus LMB. Before cell death, the levels of tyrosine phosphorylation returned to those of untreated cells. Thus, the precipitous death could be correlated with the recovery of Bcr-Abl kinase activity.

Recent studies have shown that infection of mouse bone marrow cells with a Bcr-Abl retrovirus can cause a CML-like syndrome following transplantation of infected cells into syngeneic mice (Daley et al., 1990; Li et al., 1999; Pear et al., 1998; Zhang et al., 1998). As an ex vivo verification of the results obtained with cell lines, STI571 and LMB were tested for their ability to kill Bcr-Abl-infected primary bone marrow cells. Mouse bone marrow cells were collected 5 days after 5-fluoracil (5-FU) injection. Cells were infected with a retrovirus that expresses the p210 Bcr-Abl and the green fluorescent protein (GFP) from a single bi-cistronic RNA (Zhang et al., 1998). The Bcr-Abl-infected cells could therefore be followed as GFP⁺ cells. Mixed populations of GFP⁺ and GFP⁻ bone marrow cells were treated with STI and LMB either alone or in combination for a total of 12 hours, washed and cultured the cells for 60 hours. The percentage of GFP⁺ cells were determined by FACS (FIG. 4). Before treatment (BT), 10-12% of the population was positive for GFP. In the untreated populations (NT), the GFP⁺ cells remained at 10-12%. Treatment with LMB (L) or STI571 (S) alone resulted in a moderate increase in the percent of GFP⁺ cells. However, the combined treatment with LMB and STI571 (L+S) reduced the GFP⁺ cells to a level of 1-2%. These experiments demonstrate that the treatment regimen is useful for the killing of Bcr-Abl cells ex vivo as well as in vitro.

A similar experiment was performed with bone marrow cells isolated from mice that had not been injected with 5-FU (FIG. 4 b). Before treatment, 18-20% of the cells were positive for GFP (BT). Treatment with LMB or STI571 alone had no significant effect on the percentage of GFP⁺ cells (FIG. 4 b). The combined treatment with LMB and STI571 again reduced the GFP⁺ cells to 1-2% (FIG. 4 b). The total number of GFP⁻ and GFP⁺ cells were determined in these populations (FIG. 4 c). Without drug treatment, both GFP⁻ and GFP⁺ cells increased by 40-50% relative to the starting populations (FIG. 4 c, NT). LMB exhibited a toxic effect by reducing both the GFP⁻ and GFP⁺ cells to a similar level (FIG. 4 c, L). STI571 was also toxic to both the GFP⁻ and GFP⁺ cells (FIG. 4 c, S). The mouse bone marrow cells were cultured in media containing the stem-cell factor, which acts through the c-Kit receptor tyrosine kinase. Because STI571 can inhibit the c-Kit tyrosine kinase (Heinrich et al., 2000), the sensitivity of GFP⁻ cells to STI571 could be due to the interference of c-Kit function. The combined treatment with STI571 and LMB was somewhat more toxic to the GFP⁻ cells then either drug alone (FIG. 4 c). However, the combined treatment with STI571 and LMB removed the majority (>97%) of the GFP⁺ cells (FIG. c). Thus, primary bone marrow cells that had been infected with a retrovirus to express the Bcr-Abl tyrosine kinase could be preferentially eliminated by the combined treatment with LMB and STI571.

EXAMPLE 1

Transfection and Immunoblotting. Transfection experiments were performed using the calcium phosphate method. Immunoblots were performed using 1 μg/ml 4G10 monoclonal antibody against phosphotyrosine (Upstate Biotechnology, Waltham, Mass.), or 1 μg/ml monoclonal antibody against Abl (8E9) (Pharmingen, San Diego, Calif.). All immunoblots were revealed by enhanced chemiluminescence (Amersham, Little Chalfont, UK).

EXAMPLE 2

Plasmids. The murine Abl-NES and p190 Bcr-Abl-KD have been described (Cortez et al., 1995; McWhirter and Wang, 1993; and Taagpera, et al., 1998). The BcrAbl-NES was engineered by joining the first 1527 base pairs of human Bcr with the last 3117 base pairs of murine Abl-NES.

EXAMPLE 3

Immunofluorescence. Abl^(−/−) fibroblasts were seeded onto cover slips, transfected with the specified expression plasmids and fixed for immunofluorescence 48 h after transfection. The non-adherent TonB210 and K562 cells were seeded onto poly-L-lysine (Sigma) coated cover slips for 3 h and then processed for immunofluorescence. Cells were permeabilized for 10 min with 0.3% Triton X-100 (Sigma), then blocked at room temperature (RT) for 45 to 60 min with 10% normal goat serum (Gibco, Rockville, Md.), and incubated at RT with anti-Abl 8E9 (30 μg/mL) for 60 to 90 min. Cover slips were incubated at RT for 45 to 60 min with Alexa Fluor 594 goat secondary antibody against mouse (Molecular Probes, Eugene, Oreg.) and with Phalloidin-conjugated Alexa Fluor 488 (Molecular Probes) to stain for F-Actin. After DNA staining with Hoechst 33258 (Sigma), cover slips were mounted onto glass slides with gel mount (Biomeda, Foster City, Calif.). Epifluorescence microscopy was performed with a Nikon microscope and two-dimensional images were digitally acquired with a 0.60X HRD060-NIK CCD-Camera (Diagnostic Instruments, Sterling Heights, Mich.). Deconvolution microscopy was performed with a Delta Vision System. At least 40 optical sections (0.2 microns) were collected for each cell. Quantification of nuclear signal was performed with the Image-Pro Plus 3.0 software (Media Cybernetics, Silver Spring, Md.). For each sample, 40 images in 2 dimensions and 4 images in 3 dimensions were acquired. Nuclear intensity (single pixel intensity×total number of pixels per nucleus) and cytoplasmic intensity (single pixel intensity number of pixels per cell−nuclear intensity) were calculated. Values obtained from images acquired in two dimensions (40 cells±s.d.) were corrected for cytoplasmic contamination of nuclear signal based on the parameters obtained from deconvolution microscopy.

EXAMPLE 4

Cell Death Assays. TUNEL assays were performed with the apoptosis detection system, Fluorescein (Promega) according to the manufacturer's protocol. Transfected cells were treated with STI571 alone for 12 h followed by LMB plus STI571 for another 12 h. The STI571 was then washed away and cells were kept in LMB for another 12 h to allow the recovery of Bcr-Abl kinase activity in the nucleus. TUNEL-positive cells were scored from both Bcr-Abl-expressing and untransfected cells on the same coverslip with at least 30 TUNEL-positive cells counted per experiment.

Annexin V binding to phosphatidylserine was measured with the Vibrant apoptosis assay kit #2 (Molecular Probes) following the manufacturer's recommendations. Cells were analyzed with a FACSCalibur (Becton Dickinson, Franklin Lakes, N.J.) flow cytometer. Caspase-3 activity was measured with the EnzCheck caspase-3 assay kit #1 (Molecular Probes) according to the manufacturer's protocol. Fluorescence emission was then detected at 450 nM with a Spectramax Gemini Dual Scanning Microplate Spectrofluorometer (Molecular Devices, Sunnyvale, Calif.).

EXAMPLE 5

Cell Survival Assay. 2×10⁶ TonB, TonB210 or K562 cells were plated and either left untreated or exposed to LMB, STI571, or a combination of the two drugs. After 48 h (TonB, TonB210) or 72 h (K562), live cells were counted with the Trypan blue exclusion assay. Cells were then washed extensively with PBS and placed in growth media. Trypan blue cell counts were repeated every 48 h and followed by feeding with fresh media. Cell counts were stopped when the number of viable cells was higher than the number of cells originally plated.

EXAMPLE 6

Specific killing of Bcr-Abl expressing cells in primary mouse bone marrow cells. Bone marrow was collected from mice as described (Li et al., 1991) with minor modifications. 6-wk-old Balb/c mice (Jackson, Bar Harbor, Me.) were either injected (IV) with 5-FU (5 mg) or left untreated. After 5 days, the mice were killed and their bone marrow was collected under sterile conditions. After 24 h, cells were infected with the supernatant of Bosc23 cells (Pear et al., 1993) transfected two days earlier with murine stem cell retrovirus (MSCV)-Bcr-Abl/p210-IRES-GFP (Zhang et al., 1998). Retroviral infection was carried out by cosedimentation at 1000 g for 90 min in a Sorvall RT-7 centrifuge (Sorvall, Newtown, Conn.). Medium was changed after 5 hours of adsorption and the next day the cells were subjected to a second round of retroviral transduction. The mixed GFP⁺ and GFP⁻ cell population was divided into 4 treatment conditions: untreated, exposed to 10 nM LMB for 8 h, 10 μM STI571 for 12 h or a combined treatment of 10 μM STI571 for 12 h period with 10 nM LMB added for the last 8 h. At the end of the 12-hour period, the cells were replated in prestimulation medium and cultured for 60 h. Cells were then collected and analyzed with a FACSCalibur fluorescent cell sorter (Becton Dickinson) to determine the number of GFP⁻ and GFP⁺ cells.

EXAMPLE 7

Treatment of CML with STI571-LMB combined therapy. Patients with CML in the chronic phase, defined by the presence of less than 15 percent blasts or basophils in the peripheral blood, who test positive for the Philadelphia chromosome are considered eligible for such therapy. Baseline lab values, including blood cell counts, are obtained before the initiation of therapy. Patients are treated for two to four days with a combination of STI571 at a dose of 500-1000 pg per day, administered orally, and LMB at a dose of 50-2000 pg per day administered intravenously. After the initial treatment with both drugs, administration of STI571 is discontinued.

Administration of LMB is continued for 3-5 days at the same or reduced level. Approximately one week after the course of treatment is completed, laboratory values are again obtained to determine the efficacy of treatment. The regimen is repeated as required at 1 to 3 month intervals until disease is eradicated.

EXAMPLE 8

Treatment of patients in blast crisis phase of CML. Treatment regimens of patients in blast crisis phase of CML is essentially identical to the treatment method of patients in the chronic phase. As only a portion of the cellular Bcr-Abl needs to be translocated into the nucleus for the therapy to work, higher doses of the drugs should not be required. Patients are treated concurrently with the two drugs for 2 to 4 days, followed by treatment with LMB alone for 3-5 days. Efficacy of the course of treatment is evaluated approximately one week after it is completed. The regimen may be repeated and/or combined with purging of bone marrow ex vivo as described in Example 10.

EXAMPLE 9

Treatment of solid tumors with gene transfer of NES-defective Bcr-Abl and STI571-LMB combined therapy. Bcr-Abl does not induce the formation of solid tumors; therefore, therapy comprising administration of 300-1000 μg/day of STI571 and LMB would not be useful in the treatment of such tumors. However, the introduction of “suicide genes” into tumors to cause them to become susceptible to drug treatments. The adenoviral EI B mutated vector of U.S. Pat. No. 6,080,578 can readily be modified to express a nuclear export defective Bcr-Abl. The adenovirus is injected into the tumor. Two to five days after injection, the patient is treated with a combination of STI571 (300-1000 μg per day) and LMB (50-2000 μg per day) for 2 to 4 days. As the Bcr-Abl does not contain a functional nuclear export signal, it is not necessary to maintain the patient on LMB after the initial administration of the LMB. Approximately a week after treatment, the patient is monitored for changes in tumor size and other properties.

EXAMPLE 10

Treatment of solid tumors with a nuclear targeted Bcr-Abl. A modified brc-abl construct has been generated that contains a mutated nuclear export signal and three SV40 large T antigen nuclear localization signals (SEQ ID NO 3, 4). The modified Bcr-Abl protein localizes to the nucleus in the absence of drug. An adenoviral associated vector carrying the coding sequence for this protein is injected directly into solid tumors. The protein is expressed in the nucleus and induces apoptosis killing the cancer cells in the tumor.

EXAMPLE 11

Autologous bone marrow transplant for the treatment of CML. Bone marrow is extracted from a patient with CML and transferred into culture ex vivo. Cells are purged of Bcr-Abl expressing cells by treatment with a kinase inhibitor and a nuclear export inhibitor together, followed by treatment with the nuclear export inhibitor alone. Cells are treated with a combination of a final concentration of 0.01-50 μM, preferably 1 pM, STI571 and 0.1-500 nM, preferably 10 nM LMB for 8 to 48 hours, preferably 12 hours, with LMB alone added for the last 4-48 hours, preferably 12 hours. Purged cells are then washed, possibly expanded, and prepared for transfusion back into the patient. During the time that the bone marrow cells are being purged, the patient is treated with full body radiation to kill the leukemic cells. Subsequently the purged bone marrow is returned to the patient and the disease state is monitored.

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Although an exemplary embodiment of the invention has been described above by way of example only, it will be understood by those skilled in the field that modifications may be made to the disclosed embodiment without departing from the scope of the invention, which is defined by the appended claims. 

1. A method for treating leukemia, the method comprising: a) contacting a population of bone marrow cells with an apoptosis-inducing amount of a combination of a kinase inhibitor and a nuclear export inhibitor; and b) subsequently contacting the population of bone marrow cells with the nuclear export inhibitor not in combination with the kinase inhibitor.
 2. A method according to claim 1, wherein the population of bone marrow cells is a mixture comprising leukemic and non-leukemic bone marrow cells, and wherein the kinase inhibitor and the nuclear export inhibitor combination followed by the nuclear export inhibitor not in combination with the kinase inhibitor are contacted in amounts sufficient to induce apoptosis in the leukemic bone marrow cells and not sufficient to induce apoptosis in the non-leukemic bone marrow cells.
 3. A method according to claim 2, wherein substantially all of the leukemic bone marrow cells express a Bcr-Abl protein.
 4. A method according to claim 1, wherein the kinase inhibitor is capable of inhibiting c-Abl.
 5. A method according to claim 1, wherein the kinase inhibitor is selected from the group consisting of STI571, PD173955 and PD180970.
 6. A method according to claim 1, wherein the kinase inhibitor is STI571.
 7. A method according to claim 1, wherein the nuclear export inhibitor is Leptomycin B.
 8. A method according to claim 1, wherein the leukemia is a chronic myologenous leukemia.
 9. A method according to claim 1, wherein the population of bone marrow cells is isolated from a mammal.
 10. A method according to claim 9, wherein the mammal is a mouse.
 11. A method according to claim 9, further comprising: c) administering at least one bone marrow cell to the mammal.
 12. A method according to claim 9, further comprising: c) killing substantially all leukemic bone marrow cells comprised by the mammal; and d) subsequently administering at least one bone marrow cell to the mammal.
 13. A method according to claim 1, wherein the population of bone marrow cells is comprised by a mammal.
 14. A method according to claim 13, wherein the mammal is a mouse.
 15. A method for selectively inducing apoptosis in leukemic bone marrow cells, wherein the leukemic bone marrow cells are comprised by a mixture of leukemic and non-leukemic bone marrow cells, the method comprising: a) contacting a combination of a kinase inhibitor and a nuclear transport inhibitor with the mixture of leukemic and non-leukemic bone marrow cells; and b) subsequently contacting the mixture of leukemic and non-leukemic bone marrow cells with the nuclear export inhibitor not in combination with the kinase inhibitor, wherein the kinase inhibitor and a nuclear transport inhibitor combination followed by the nuclear export inhibitor not in combination with the kinase inhibitor is contacted in an amount sufficient to induce apoptosis in the leukemic bone marrow cells and not sufficient to induce apoptosis in the non-leukemic bone marrow cells.
 16. A method according to claim 15, wherein substantially all of the leukemic bone marrow cells comprise a Bcr-Abl protein.
 17. A method according to claim 15, wherein the kinase inhibitor is capable of inhibiting c-Abl.
 18. A method according to claim 15, wherein the kinase inhibitor is selected from the group consisting of STI571, PD173955 and PD180970.
 19. A method according to claim 15, wherein the kinase inhibitor is STI571.
 20. A method according to claim 15, wherein the nuclear export inhibitor is Leptomycin B.
 21. A method according to claim 15, wherein the mixture of leukemic and non-leukemic bone marrow cells is isolated from a mammal.
 22. A method according to claim 21, wherein the mammal is a mouse.
 23. A method according to claim 15, wherein the mixture of leukemic and non-leukemic bone marrow cells is comprised by a mammal.
 24. A method according to claim 23, wherein the mammal is a mouse.
 25. A method according to claim 1, wherein the population of bone marrow cells is a mixture comprising leukemic and non-leukemic bone marrow cells, and wherein the kinase inhibitor and the nuclear export inhibitor combination followed by the nuclear export inhibitor not in combination with the kinase inhibitor are contacted in amounts sufficient to induce apoptosis in the leukemic bone marrow cells and not sufficient to induce apoptosis in the non-leukemic bone marrow cells, and further wherein the kinase inhibitor is STI571 and the nuclear export inhibitor is Leptomycin B.
 26. A method of claim 25 wherein the leukemic cells comprise CML or ALL.
 27. A method according to claim 1, wherein the population of bone marrow cells is a mixture comprising leukemic and non-leukemic bone marrow cells, and wherein the kinase inhibitor and the nuclear export inhibitor combination followed by the nuclear export inhibitor not in combination with the kinase inhibitor are contacted in amounts sufficient to induce apoptosis in the leukemic bone marrow cells and not sufficient to induce apoptosis in the non-leukemic bone marrow cells, and further wherein the kinase inhibitor is PD173955 and PD180970, and the nuclear export inhibitor is Leptomycin B.
 28. A method of claim 27 wherein the leukemic cells comprise CML or ALL 